Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency

J. C M Van Der Loo, W. P. Swaney, E. Grassman, A. Terwilliger, T. Higashimoto, A. Schambach, S. Hacein-Bey-Abina, D. L. Nordling, M. Cavazzana-Calvo, A. J. Thrasher, D. A. Williams, L. Reeves, P. Malik

Research output: Contribution to journalArticle

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Abstract

Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68-70% normal human CD34 + cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdc scid/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34 +cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.

Original languageEnglish
Pages (from-to)872-876
Number of pages5
JournalGene Therapy
Volume19
Issue number8
DOIs
StatePublished - Aug 2012

Fingerprint

X-Linked Combined Immunodeficiency Diseases
Severe Combined Immunodeficiency
Genetic Therapy
Gibbon ape leukemia virus
Interleukin Receptor Common gamma Subunit
Colony-Forming Units Assay
Heterologous Transplantation
Proto-Oncogenes
Terminal Repeat Sequences
Product Packaging
Therapeutics
Glutamine
Interleukin-2
Transfection
Culture Media
Cations
Buffers
Leukemia
T-Lymphocytes
Safety

Keywords

  • clinical-grade
  • gamma-retrovirus
  • GMP
  • process development
  • SCID-X1
  • self-inactivating

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Van Der Loo, J. C. M., Swaney, W. P., Grassman, E., Terwilliger, A., Higashimoto, T., Schambach, A., ... Malik, P. (2012). Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency. Gene Therapy, 19(8), 872-876. https://doi.org/10.1038/gt.2012.37

Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency. / Van Der Loo, J. C M; Swaney, W. P.; Grassman, E.; Terwilliger, A.; Higashimoto, T.; Schambach, A.; Hacein-Bey-Abina, S.; Nordling, D. L.; Cavazzana-Calvo, M.; Thrasher, A. J.; Williams, D. A.; Reeves, L.; Malik, P.

In: Gene Therapy, Vol. 19, No. 8, 08.2012, p. 872-876.

Research output: Contribution to journalArticle

Van Der Loo, JCM, Swaney, WP, Grassman, E, Terwilliger, A, Higashimoto, T, Schambach, A, Hacein-Bey-Abina, S, Nordling, DL, Cavazzana-Calvo, M, Thrasher, AJ, Williams, DA, Reeves, L & Malik, P 2012, 'Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency', Gene Therapy, vol. 19, no. 8, pp. 872-876. https://doi.org/10.1038/gt.2012.37
Van Der Loo, J. C M ; Swaney, W. P. ; Grassman, E. ; Terwilliger, A. ; Higashimoto, T. ; Schambach, A. ; Hacein-Bey-Abina, S. ; Nordling, D. L. ; Cavazzana-Calvo, M. ; Thrasher, A. J. ; Williams, D. A. ; Reeves, L. ; Malik, P. / Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency. In: Gene Therapy. 2012 ; Vol. 19, No. 8. pp. 872-876.
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